The present invention relates to a novel quaternary ammonium salt and a process for the preparation thereof. The compound according to the present invention is useful as an antimicrobial agent such as preservative for eye drop.
A quaternary ammonium salt having an antimicrobial activity has long been known and is now widely used. However, conventional quaternary ammonium salts are normally disadvantageous in that they show deteriorated sterilizing power and antimicrobial power due to the effect of saccharides, protein and lipid or in an acid range having a low pH value and have no effect on cellular spore.
As solutions to the foregoing problems, a compound having two structural units called quaternary ammonium per molecular has been proposed, which unit is capable of having four alkyl groups or other groups connected to one nitrogen atom, e.g., the antimicrobial agent comprising quaternary ammonium salt in JP-A-6-321902 (The term xe2x80x9cJP-Axe2x80x9d as used herein means an xe2x80x9cunexamined published Japanese patent applicationxe2x80x9d) and JP-A-10-114604. The compound disclosed in these published applications has methyl or ethyl groups connected thereto in a proportion of 2 or more per one of nitrogen atoms constituting the quaternary ammonium. Accordingly, this compound has a high antimicrobial activity and thus is a good compound from the standpoint of antimicrobial properties. This compound has been actually used as an antimicrobial agent. This compound has a high antimicrobial activity but it has been desired to improve the safety of this compound to human.
It is therefore an object of the present invention to provide a novel compound which exhibits a high antimicrobial activity and a wide antimicrobial spectrum and can be used as an antimicrobial agent having a high safety to human.
The other objects of the present invention will become apparent from the following detailed description and examples.
The present invention lies in a quaternary ammonium salt represented by the following general formula (1): 
wherein R1 and R2 each represent a C4-12 alkyl group and may be the same or different; R3 represents a C1-2 alkyl group; k represents an integer of from 0 to 4; X represents an inorganic or organic anion group; n represents the valence of the anion group X which is selected from 1 and 2; and m represents an integer of 2 when n is 1 or 1 when n is 2.
The compound of the invention can be obtained by the following reaction [I], [II] or [III]:
Reaction [I]
In the general formula (6), k represents an integer of from 0 to 4, and Y represents a chlorine atom, bromine atom or iodine atom.
In the general formula (7), R1 and R2 each represent a C4-12 alkyl group, and R3 represents a C1-2 alkyl group.
Reaction [II]
In the general formula (8), R1 and R2 may be the same or different and each represent a C4-12 alkyl group, and k represents an integer of from 0 to 4.
In the general formula (9), R3 represents a C1-2 alkyl group, and Z represents a chlorine atom, bromine atom, iodine atom or a group represented by any one of the following general formulae (10) and (11): 
wherein R6 represents a C1-2 alkyl group; and 
wherein R7 and R8 each represent a hydrogen atom, C1-2 alkyl group or carboxyl group.
Reaction [III]: 
In the general formula (12) R1 represents a C4-12 alkyl group, R3 represents a C1-2 alkyl group, and k represents an integer of from 0 to 4.
In the general formula (13), R2 represents a C4-12 alkyl group; and J represents a chlorine atom, bromine atom, iodine atom or a group represented by the general formula (11).
A. Novel Compound
The present invention relates to a novel quaternary ammonium salt represented by the following general formula (1): 
wherein R1 and R2 each represent a C4-12 alkyl group and may be the same or different; R3 represents a C1-2 alkyl group; k represents an integer of from 0 to 4; X represents an inorganic or organic anion group; n represents the valence of the anion group X which is selected from 1 and 2; and m represents an integer of 2 when n is 1 or 1 when n is 2.
In the general formula (1), the alkyl group represented by R1 or R2 there may be used an alkyl group having from 4 to 12 carbon atoms. In order to balance between antimicrobial properties and safety to human, the number of carbon atoms in the alkyl group is preferably from 5 to 10, more preferably from 6 to 8. R3 may be either methyl or ethyl but is preferably methyl to exhibit higher antimicrobial properties.
In the general formula (1), X represents an inorganic or organic anion group. Preferred examples of such an inorganic or organic anion group include iodine ion, bromine ion, chlorine ion, fluorine ion, iodic acid ion, bromic acid ion, chloric acid ion, periodic acid ion, perchloric acid ion, chlorous acid ion, hypochlorous acid ion, nitric acid ion, nitrous acid ion, sulfuric acid ion, hydroxyl group ion or an anion group represented by any one of the following general formulae (2) to (5).
The number (m) of anion groups X connected to the compound of the general formula (1) is such that the product of n and m is 2 wherein n is the valence of the anion group X. For example, when the valence of the anion group X is xe2x88x922, m is 1. When the valence of the anion group X is xe2x88x921, m is 2.
[R4COOxe2x8ax96]xe2x80x83xe2x80x83(2)
wherein R4 represents a C1-7 alkyl or alkenyl group which may have one or more hydroxyl group or carbonyl group;
xe2x80x83[xe2x8ax96OCOxe2x80x94R5xe2x80x94COOxe2x8ax96]xe2x80x83xe2x80x83(3)
wherein R5 does not exist (the two groups COO are directly bonded to each other) or represents a C1-8 alkyl or alkenyl group which may have one or more hydroxyl group;
[R6SO4xe2x8ax96]xe2x80x83xe2x80x83(4)
wherein R6 represents a C1-2 alkyl group; and 
wherein R7 and R8 each represent a hydrogen atom, C1-12 alkyl group or carboxyl group.
B. Process for the Preparation of Novel Compound
The quaternary ammonium salt represented by the general formula (1) can be prepared by any one of the following processes.
B-1. First-class Preparation Process
The first embodiment of the process for the preparation of the quaternary ammonium salt represented by the general formula (1) involves the reaction of a halogen compound represented by the following general formula (6) with a tertiary amine represented by the following general formula (7): 
wherein k represents an integer of from 0 to 4; and Y represents a chlorine atom, chlorine atom or iodine atom; and 
wherein R1 and R2 each represent a C4-12 alkyl group; and R3 represents a C1-2 alkyl group.
Examples of the compound represented by the general formula (6) include halogen compounds such as xcex1, xcex1xe2x80x2-dichloro-o-xylene, xcex1, xcex1xe2x80x2-dichloro-m-xylene, xcex1, xcex1xe2x80x2-dichloro-p-xylene, xcex1, xcex1xe2x80x2-dibromo-o-xylene, xcex1, xcex1xe2x80x2-diodo-m-xylene, 3,6-bis(chloromethyl)durene, 2,4-bis(chlorometh)-dimethylbenzene and 2,5-bis(chloromethyl)toluene.
Examples of the compound represented by the general formula (7) include tertiary amines such as N,N-dipentyl-N-methylamine, N,N-dihexyl-N-methylamine, N,N-diheptyl-N-methylamine, N-butyl-N-hexyl-N-methylamine, N-pentyl-N-hexyl-N-methylamine, N-hexyl-N-octyl-N-methylamine, N,N-dipentyl-N-ethylamine, N,N-dihexyl-N-ethylamine and N-pentyl-N-hexyl-N-ethylamine.
These reactions can be effected at a temperature of from 50xc2x0 C. to 120xc2x0 C. in a proper organic solvent. The proportion of the tertiary amine of the general formula (7) to the halogen compound of the general formula (6) may be not less than 2 mols, e.g., from 2.0 to 2.3 mols per mol of the compound of the general formula (6).
As a reaction solvent there may be used an alcohol such as methanol, ethanol and n-propanol, mixture of water and alcohol or aromatic organic solvent such as benzene, toluene and xylene. The reaction temperature may be not lower than 80xc2x0 C. At this reaction temperature, the reaction can be normally completed in 1 to 40 hours.
B-2. Second-class Preparation Process
The second embodiment of the process for the preparation of the quaternary ammonium salt represented by the general formula (1) involves the reaction of a tertiary amine represented by the following general formula (8) with a quaterizing agent represented by the following general formula (9): 
wherein R1 and R2 may be the same or different and each represent a C4-12 alkyl group; and k represents an integer of from 0 to 4; and
R3xe2x80x94Z xe2x80x83xe2x80x83(9)
wherein R3 represents a C1-2 alkyl group; and Z represents a chlorine atom, bromine atom, iodine atom or a group represented by any one of the following general formulae (10) and (11): 
wherein R6 represents a C1-2 alkyl group; and 
wherein R7 and R8 each represent a hydrogen atom, C1-12 alkyl group or carboxyl group.
Examples of the compound of the general formula (8) include N,N,Nxe2x80x2,Nxe2x80x2-tetrapentyl-o-xylylenediamine, N,Nxe2x80x2-dibutyl-N,Nxe2x80x2-dihexyl-o-xylylenediamine, N,N,Nxe2x80x2,Nxe2x80x2-tetrahexyl-m-xylylenediamine, N,N,Nxe2x80x2,Nxe2x80x2-tetraheptyl-p-xlylenediamine, N,N,Nxe2x80x2,Nxe2x80x2-tetrahexyl-2,3,5,6-tetramethyl-p-xylylenediamine, N,Nxe2x80x2-dibutyl-N,Nxe2x80x2-dihexyl-2,3,5,6-tetramethyl-p-xylylenediamine, N,Nxe2x80x2-dihexyl-N,Nxe2x80x2-dioctyl-2,3,5,6-tetramethyl-p-xylylenediamine, N,N,Nxe2x80x2,Nxe2x80x2-tetrapentyl-1,3,5-trimethyl-m-xylylenediamine, N,N,Nxe2x80x2,Nxe2x80x2-tetrahexyl-2,4-dimethyl-p-xylylenediamine, and N,N,Nxe2x80x2,Nxe2x80x2-tetrahexyl-2-methyl-p-xylylenediamine.
Examples of the compound represented by the general formula (9) include halogenated alkyl such as methane chloride, ethane chloride, methane bromide, ethane bromide and methane iodide, dialkylsulfuric acid such as dimethylsulfuric acid, and sulfonic acid alkyl such as methyl p-toluenesulfonate.
These reactions can be conducted at a temperature of from 50xc2x0 C. to 120xc2x0 C. in a proper solvent. Many quaterizing agents are inactivated when acted upon by heat or the like. The quaterizing agent compound represented by the general formula (9) is preferably used in excess with the tertiary amine represented by the general formula (8), i.e., not lower than 4 mols, more preferably not lower than 6 mols per mol of the tertiary amine represented by the general formula (8).
Examples of the reaction solvent employable herein include alcohol such as methanol, ethanol, n-propanol and 2-methoxy-ethanol, and mixture of water and alcohol. Further examples of the reaction solvent include aprotic solvent such as N,N-dimethylformamide, N-methylformamide, nitromethane, nitroethane, and acetonitrile.
Referring to the reaction atmosphere, synthesis can be made in the atmosphere. However, the reaction is preferably effected in a nitrogen atmosphere. The reaction temperature may be not lower than 80xc2x0 C. At this temperature, the reaction can be normally completed in 1 to 40 hours.
The foregoing reaction may also be effected at a temperature of from 50xc2x0 C. to 100xc2x0 C. under pressure, preferably from 10 to 100 MPa (megapascal) in a proper solvent in an autoclave. The reaction time may be normally from 5 hours to 120 hours.
The tertiary amine of the general formula (8) can be obtained, e.g, by the reaction of the halogen compound of the general formula (6) with a compound represented by the following general formula (15): 
wherein R1 and R2 each represent a C4-12 alkyl group.
Examples of the compound of the general formula (15) include secondary amines such as N,N-dipentylamine, N,N-dihexylamine, N-butyl-N-hexylamine and N-butyl-N-octylamine.
The tertiarization reaction of the compound of the general formula (6) with the compound of the general formula (15) may be effected at a temperature of from 50xc2x0 C. to 120xc2x0 C. in a proper organic solvent such as alcohol, mixture of alcohol and water and aromatic organic solvent for 1 hour to 48 hours. The proportion of the secondary amine of the general formula (15) in the halogen compound of the general formula (6) is preferably 4 to 6 mols, particularly from 4.1 to 4.4 mols per mol of the compound of the general formula (6). As a result of the foregoing tertiarization reaction, a tertiary amine and a hydrochloride of amine are produced as a desired intermediate product and a by-product which is used as a starting material, respectively.
The separation of the tertiary amine as a desired intermediate product and the hydrochloride of amine as a starting material may be carried out by any method such as extraction. Further, the separation of the tertiary amine and the hydrochloride of amine can be easily accomplished by the use of the difference in solubility between organic solvents.
The hydrochloride of amine as a by-product can be regenerated to a starting material amine. When the hydrochloride of amine as a by-product is acted upon by a base such as aqueous solution of NaOH, a water-insoluble organic phase is produced. When the organic phase is subjected to ordinary separation/purification, a secondary amine is obtained as a starting material. The secondary amine thus obtained can be again used as a starting material.
B-3. Third-class Preparation Process
The third embodiment of the process enabling the preparation of the quaternary ammonium salt represented by the general formula (1) involves the treatment of a tertiary amine represented by the following general formula (12) with a quaterizing agent represented by the following general formula (13): 
wherein R1 represents a C4-12 alkyl group; R3 represents a C1-2 alkyl group; and k represents an integer of from 0 to 4.
R2xe2x80x94J xe2x80x83xe2x80x83(13)
wherein R2 represents a C4-12 alkyl group; and J represents a chlorine atom, bromine atom, iodine atom or a group represented by the general formula (11).
Examples of the compound represented by the general formula (12) include N,Nxe2x80x2-dipentyl-N,Nxe2x80x2-dimethyl-o-xylylenediamine, N,Nxe2x80x2-dihexyl-N,Nxe2x80x2-dimethyl-o-xylylene diamine, N,Nxe2x80x2-dipentyl-N,Nxe2x80x2-diethyl-o-xylylenediamine, N,Nxe2x80x2-dipentyl-N,Nxe2x80x2-dimethyl-m-xylylenediamine, N,Nxe2x80x2-dipentyl-N,Nxe2x80x2-dimethyl-p-xylylenediamine, N,Nxe2x80x2-dihexyl-N,Nxe2x80x2-dimethyl-2,3,5,6-tetramethyl-p-xylylenediamine, N,Nxe2x80x2-dipentyl-N,Nxe2x80x2-dimethyl-1,3,5-timethyl-m-xylylenediamine, and N,Nxe2x80x2-dihexyl-N,Nxe2x80x2-dimethyl-2,4-dimethyl-p-xylylenediamine.
Examples of the compound represented by the general formula (13) include halogenated alkyl such as butane chloride, pentane chloride, hexane chloride, decane chloride, nonane bromide, decane bromide, pentane iodide and heptane iodide, and sulfonic acid alkyl such as butyl p-toluenesulfonate, pentyl p-toluenesulfonate, hexane p-toluenesulfonate and dodecane p-toluenesulfonate.
The reaction of the compound of the general formula (12) with the compound of the general formula (13) can be effected substantially according to the second preparation process. In some detail, as a solvent there may be preferably used an alcohol, mixture of water and alcohol or aprotic solvent such as N,N-dimethylformamide and methyl cellosolve. The reaction can be effected in the absence of solvent. The reaction atmosphere, reaction temperature and reaction time may be the same as used in the second preparation process. The proportion of the quaterizing agent of the general formula (13) in the tertiary amine of the general formula (12) may be not smaller than 2 mols, e.g., from 2.0 to 2.3 mols per mol of the compound of the general formula (12). The foregoing reaction can proceed in the presence of a proper solvent under pressure in an autoclave in the same manner as in the second reaction.
The tertiary amine represented by the general formula (12) can be obtained by the reaction of the halogen compound represented by the general formula (6) with a compound represented by the following general formula (16): 
wherein R1 represents a C4-12 alkyl group; and R3 represents a C1-2 alkyl group.
Examples of the compound represented by the general formula (16) include secondary amines such as N-pentyl-N-methylamine, N-hexyl-N-methylamine and N-hexyl-N-ethylamine.
The tertiarization reaction of the compound represented by the general formula (6) with the compound represented by the general formula (16) can be effected at a temperature of from 50xc2x0 C. to 120xc2x0 C. in a proper organic solvent such as alcohol, mixture of water and alcohol and aromatic organic solvent for 1 hour to 48 hours substantially according to the reaction of the compound represented by the general formula (6) with the compound represented by the general formula (15). The proportion of the secondary amine represented by the general formula (16) in the halogen compound represented by the general formula (6) is preferably from 4 to 6 mols, particularly from 4.1 to 4.4 mols per mol of the compound of the general formula (6). The separation of the tertiary amine as a desired intermediate product and the hydrochloride of amine as a starting material and the regeneration of the starting material amine hydrochloride as a by-product can be effected substantially according to the reaction of the compound represented by the general formula (6) with the compound represented by the general formula (15).
B-4. Process for the Purification of Compound
The compound produced by the foregoing third preparation process can be easily purified by an ordinary separation/purification method such as column chromatographic separation and recrystallization as necessary.
B-5. Exchange of Anion Group
The quaternary ammonium salt prepared by the foregoing third preparation process can be subjected to ion exchange to exchange the anion group incorporated therewith with another specific anion group. The ion exchange can be easily carried out by treating the quaternary ammonium salt in a column filled with an anion exchange resin.
In some detail, the quaternary ammonium salt of the invention comprises as a cation group a quaternary ammonium salt represented by the following general formula (14) incorporated therein. The anion as the counter ion of such a cation group there may be prepared by effecting ion exchange so that the anion group synthesized by any one the foregoing three preparation processes is replaced by an iodine ion (Ixe2x88x92), bromine ion (Brxe2x88x92), chlorine ion (Clxe2x88x92), iodic acid ion (IO3xe2x88x92), bromic acid ion (BrO3xe2x88x92), chloric acid ion (ClO3xe2x88x92), periodic acid ion (IO4xe2x88x92), erchloric acid ion (ClO4xe2x88x92), chlorous acid ion (ClO2xe2x88x92), hypochlorous acid ion (ClOxe2x88x92), nitric acid ion (NO3xe2x88x92), nitrous acid ion (NO2xe2x88x92), sulfuric acid ion (SO42xe2x88x92), hydroxyl group ion (OHxe2x88x92) or an anion group represented by any one of the following general formulae (2) to (5): 
wherein R1 and R2 each represent a C4-12 alkyl group and may be the same or different; R3 represents a C1-2 alkyl group; and k represents an integer of from 0 to 4.
C. Antimicrobial Properties
The compound of the invention represented by the general formula (1) thus obtained exhibits a wide antimicrobial spectrum against various bacteria and fungi as described later in Test Examples 1 and 2.
The compound of the invention represented by the general formula (1) exhibits a particularly strong sterilizing activity when the alkyl chain (R1, R2) has from 5 to 10, particularly from 6 to 8 carbon atoms.
The compound of the invention represented by the general formula (1) exhibits an excellent sterilizing activity, i.e., minimum sterilizing concentration of from {fraction (1/10)} to {fraction (1/100)} of that of conventional commercial quaternary ammonium salts. Accordingly, the compound of the invention represented by the general formula (1) can exert a sterilizing effect equal to that of the conventional commercial sterilizers of the same kind when used in an amount far less than that of the conventional sterilizers.
D. Safety of Compound
The quaternary ammonium salt of the invention is a compound having an extremely high safety, i.e., LD50 (rat) of not smaller than 2,000 mg/kg in oral toxicity test on rat.
Further, the compound of the invention represented by the general formula (1) was determined for hemolytic activity. As a result, the compound of the invention represented by the general formula (1) was found to have a hemolytic activity of 10 times or more lower than that of conventional quaternary ammonium salts such as benzalkonium chloride and those proposed in JP-A-6-321902 and JP-A-10-114604. Thus, the compound of the invention represented by the general formula (1) exhibits an extremely low toxicity to human being.
E. Usage of Compound
The compound according to the invention can be used as an antimicrobial agent in a wide range of arts such as antimicrobial deodorized fiber product, leather product, building material, coating compound, adhesive, plastic, film, paper, pulp, metal working oil, food, pharmaceuticals, medical and atmospheric infectants, eye drop, detergent, cosmetics, stationary product, agricultural chemicals, stock farming, etc.
The compound according to the invention can exhibit an antimicrobial effect without any supplemental ingredient or with solid or liquid carrier. Depending on the situation, surface-active agent, other ingredient, etc. can be mixed to form emulsion, hydrate agent, granular agent, powder, spray agent or aerosol.
The proportion of the compound of the invention is normally from 0.0001 to 100% by weight, preferably from 0.001 to 10% by weight on the total amount of antimicrobial agent.
F. Use as Preservative for Eye Drop
Examples of conventional preservatives for eye drop include compounds having cation group such as quaternary ammonium salt and guanidine, alcohols, aminobenzoic acid ester, and sorbic acid. Quaternary ammonium salts, particularly benzalkonium chloride, are commonly used because they have a great anti-septic power.
However, when these preservatives are incorporated in an eye drop, the resulting eye drop can become slightly turbid depending on the kind of other compounds to be incorporated as pharmaceutical components. It has been reported that when benzalkonium chloride is incorporated in an amount of not smaller than 0.01%, the cornea is damaged. Thus, the amount of benzalkonium chloride to be incorporated in an eye drop is restricted to a value at which no safety problem occurs.
Thus, when the conventional preservatives are incorporated in an eye drop in a low concentration, no turbidity occurs. However, the resulting anti-septic effect is insufficient. Accordingly, various improvement techniques for enhancing anti-septic effect or preventing turbidity have been proposed as in JP-A-2-311417 and JP-A-6-40910.
The inventors made extensive studies. As a result, it was found that the compound of the invention represented by the general formula (1) (hereinafter abbreviated as xe2x80x9ccompound (1)of the inventionxe2x80x9d) is extremely effective as a preservative for providing a transparent eye drop without producing insoluble materials with various compounds used as effective components for eye drop as described later in Test Example 1.
In other words, the preservative for eye drop according to the invention comprises as an effective component the compound (1) of the invention incorporated therein.
Examples of eye drop pharmaceutical compounds which cause no turbidity even when mixed with the compound (1) of the invention include compounds having carboxyl group such as sodium hyaluronate, dipotassium glycyrrhizinate, pyrenoxin, lysozyme chloride, sodium cromoglicate and carboxy vinyl polymer, compounds having sulfonic acid group such as chondroitin sodium sulfate, sodium dimethylisopropylazulene sulfonate, sodium colistinmethanesulfonate and dexamethasone sodium metasulfobenzoate, compounds having phosphonyl group such as flavin-adenine dinucleotide, and pilocarpine hydrochloride. All these compounds have not heretofore been able to be incorporated as pharmaceutical components because they cause turbidity when mixed with benzalkonium chloride.
The anti-septic effect of the preservative for eye drop according to the invention cannot be adversely affected by various components which are optionally incorporated in an eye drop in addition to the foregoing pharmaceutical components, e.g., effective component such as antiphlogistic, vitamin and antihistamines, additives such as pH adjustor, buffer, isotonic agent and solubilizer.
Further, the compound (1) of the invention exhibits a wide sterilizing spectrum against various bacteria and fungi as well as a high sterilizing activity and anti-septic power as compared with benzalkonium chloride, which is a conventional commonly used quaternary ammonium salt, as described later in Test Examples 3 to 5.
The proportion of the compound (1) of the invention in an eye drop is normally from 0.0005 to 0.1% by weight, preferably from 0.002 to 0.02% by weight based on the total amount of the eye drop. When the proportion of the compound (1) of the invention in an eye drop falls below 0.0005% by weight, the anti-septic effect of the preservative may become insufficient. On the contrary, when the proportion of the compound (1) of the invention in an eye drop exceeds 0.1% by weight, it may be economically disadvantageous.
G. The present invention will be further described in the following Examples and Comparative Examples, but the present invention should not be construed as being limited thereto.
G-1: SYNTHESIS EXAMPLES
Examples of the synthesis of the compound of the invention will be described hereinafter.
Compounds thus synthesized will be hereinafter abbreviated as xe2x80x9cx1-BAMX2-x3x4xe2x80x9d (in which
x1 represents a number indicating the bonding position in the benzene ring in the quaternary ammonium salt (ortho position: 2; meta position: 3; para position: 4),
x2 represents a symbol corresponding to the value of k in the general formula (1) (0: p; 3: T; 4: D),
x3 represents a number of from 4 to 12 indicating the number of carbon atoms contained in the long alkyl group connected to the quaternary ammonium salt, and
x4 represents a symbol corresponding to the kind of anion (chlorine: C; bromine: B; iodine: I; sulfate: S).